Methods for inhibiting rejection of transplanted tissue

ABSTRACT

A method for inhibiting rejection by a recipient animal of a transplanted tissue, said method comprising modifying, eliminating, or masking an antigen which, when present on the surface of a cell of said tissue, is capable of causing a T-lymphocyte-mediated response in said animal, to inhibit antigen-mediated interaction between said cell and a T-lymphocyte of said animal without causing lysis of said cell.

BACKGROUND OF THE INVENTION

This application is a continuation-in-part of Faustman U.S. Ser. No.07/575,150, filed Aug. 30, 1990 now abandoned.

This invention relates to transplantation of tissues, e.g., islet cells,muscle cells, and whole organs, into hosts in need of such tissues,e.g., patients who have or are at risk of developing diabetes mellitus,have muscular dystrophy, or are in need of an organ transplant.

Diabetes mellitus is a prevalent degenerative disease, characterized byinsulin deficiency, which prevents normal regulation of blood glucoselevels, and which leads to hyperglycemia and ketoacidosis.

Insulin, a peptide hormone, promotes glucose utilization, proteinsynthesis, formation and storage of neutral lipids, and the growth ofsome cell types. Insulin is produced by the β cells within the islets ofLangerhans of the pancreas. Early-onset diabetes (10-20% of cases) iscaused by an auto-immune reaction that causes complete destruction of βcells. Adult-onset diabetes has a number of causes, but in most casesthe β islet cells are defective in secretion of insulin.

Insulin injection therapy, usually with porcine or bovine insulin,prevents severe hyperglycemia and ketoacidosis, but fails to completelynormalize blood glucose levels. While injection therapy has been quitesuccessful, it fails to prevent the premature vascular deteriorationthat is now the leading cause of morbidity among diabetics.Diabetes-related vascular deterioration, which includes bothmicrovascular degeneration and acceleration of atherosclerosis, caneventually cause renal failure, retinal deterioration, angina pectoris,myocardial infarction, peripheral neuropathy, and arteriosclerosis.

Recently, cloning of the human insulin-encoding gene has allowed largescale production of human insulin, which has begun to replace bovineinsulin and porcine insulin as the treatment of choice. Use of humaninsulin has eliminated some of the problems associated with other formsof insulin, including antibody-mediated insulin resistance and allergicreactions resulting from the slightly different structures of non-humaninsulins. Despite these advantages, treatment with human insulin doesnot prevent vascular deterioration.

Insulin delivery pumps have been developed which administer varyingdoses of insulin based on activity, diet, time of day, and otherpre-programmed factors. While such devices improve blood sugar control,they also do not prevent vascular deterioration.

Surgical transplantation of part or all of the pancreas is thought to bepotentially the best treatment for diabetes. Successful transplantationis difficult, however, because the pancreas is a fragile and complicatedorgan, and it is impossible for a human donor to give only a portion ofit; the only practicable source is a deceased donor. Further, only asmall portion of the pancreas, the β cells of the islet of Langerhans,produce insulin; the remainder of the pancreas presents a potent targetfor transplant rejection. Transplantation of just the islets ofLangerhans is a desirable goal, as they continue to secrete appropriateamounts of insulin in response to nutritional signals even when isolatedfrom the rest of the pancreas.

A major problem associated with transplantation therapy as a treatmentfor diabetes is that current regimes require life-long administration ofimmunosuppressive drugs. These drugs can cause increased susceptibilityto infection, renal failure, hypertension, and tumor growth.

Despite these serious complications, islet transplantation has beensuccessfully performed in experimental animals. Successfultransplantation in rodents has been shown to restore normal bloodglucose regulation and prevent further vascular deterioration. Thebroader application of allografts and xenografts (inter-species grafts)as a therapy for diabetes depends on preventing transplant rejection. Ithas long been known that culturing islets prior to transplantationdecreases immunogenicity and increases transplant survival (Lacy et al(1979) Science 204₋₋ 312; Lafferty et al. (1975) Science 188:259). It isthought that long term culturing removes the Ia-bearing passengerlymphoid cells, which are a primary stimulus for cell-mediated immunityand graft rejection. Faustman et al. (J. Exp. Med. 151:1563, 1980) foundthat islet cells lack Ia antigenic determinants and express class Iantigen on their surfaces. This allowed Faustman et al. (Proc. Natl.Acad. of Sci. U.S.A. 78:5156, 1981) to develop a regime that useddonor-specific anti-Ia serum and complement to destroy Ia bearinglymphoid cells in islets, and allowed transplantation across a majorhistocompatibility barrier into non-immunosuppressed diabetic mice.

SUMMARY OF THE INVENTION

The invention features a method for inhibiting rejection by a recipientanimal of a transplanted tissue. The method involves modifying,eliminating, or masking an antigen which, when present on the surface ofa cell of the tissue, is capable of causing a T-lymphocyte-mediatedresponse in the animal; modification, elimination, or masking of theantigen inhibits antigen-mediated interaction between the cell and aT-lymphocyte of the animal, without causing lysis of the cell.

Where cells of the tissue for transplantation (the "donor" tissue) bearon their surfaces HLA class I antigens (members of one of the classes ofmajor histocompatibility complex antigens), these antigens causecytotoxic T-cell activation in recipients, terminating in donor celllysis after several sequential activation steps. The cascade isinitiated by non-specific conjugate formation between the CD8 receptoron host cytotoxic T-cells and the HLA class I antigens on the donorcell. Conjugate formation is followed by T-cell-mediated lysis,resulting in donor cell death. This lytic process can result inrejection even in intraspecies transplantation. According to theinvention, this problem is addressed by masking, modifying, oreliminating of the HLA class I antigens on the donor cells, so that theCD8-HLA class I antigen interaction which initiates the lytic cascadecannot occur.

As will be explained in more detail below, any T-cellreceptor-interactive antigens on the surfaces of donor cells canadvantageously be modified, eliminated, or masked according to theinvention. The invention thus permits not just intra-speciestransplantation of tissues and organs, but xenografts as well, openingup the possibility of "farming" of donor organs and tissues in non-humananimals for transplantation into human patients.

Preferred masking agents are F(ab')₂ fragments of antibodies to donorcell antigens; these fragments, while being capable of forming immunecomplexes with the antigen and thus preventing antigen-T-cellinteraction, do not, because they have had the Fc portion of theantibody removed, fix complement and bring about cell lysis. It has beenfound that, even though one might not expect such F(ab')₂ fragments tobind tightly enough to permanently mask antigenic sites on donor cells,long-term host acceptance of such treated tissues can be achieved.

As will be explained in greater detail below, rejection-inducing surfaceantigens on cells of donor tissues can, in addition to being masked, bemodified, e.g., by "capping", or wholly or partially eliminated bygenetic manipulation, either in transgenic animals used as a source ofdonor tissue, or in culture.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The drawings are first briefly described.

Drawings

FIG. 1 is a set of graphs illustrating the expression of HLA-class I(W6/32), CD29 (4B4), CD54 (ICAM-1) and CD58 (LFA-3) on freshly isolated99-97% pure whole human islets of Langerhans by indirectimmunofluorescence and flow cytometry. A. Human islets were positive at36% with W6/32 antibody (). B. Human islets were negative for CD29 with9% expression (). C. Human islets in this clean islet preparation werevirtually negative for ICAM-1 with 14% expression (). D. Human isletswere negative for LFA-3 expression with 10.2% of the cells positive.Background goat anti-mouse FITC expression was 9% () for thisexperiment. An open gate with exclusion of dead cells and debris wasused for flow cytometry. As expected, islet preparations contaminatedwith large amounts of fibroblast overgrowth or endothelial cells (purity60-75%) were positive for low levels of LFA-3 and ICAM.

FIG. 2 is a series of photographs showing histologic analysis of humanislets transplanted under the kidney capsule of Balb/c recipients A.Photomicrograph of human islet xenograft 30 days after transplant withpretransplant treatment with HLA class I F(ab')₂ fragments (W6/32). Thisaldehyde fuscin stain (X100) shows well-granulated islets under thekidney capsule. B. Photomicrograph of human islet xenograft 200 daysafter transplant with pretransplant treatment with HLA class I F(ab')₂fragments (W6/32). This aldehyde fuscin stain (X100) showswell-granulated islets under the kidney capsule. C. A control Balb/cmouse was transplanted with untreated fresh human islets and then killedat day 30. This characteristic photomicrograph shows the absence ofdonor islets and the presence of subcapsular fibrosis demonstrating theformer area where the islets were transplanted. D. Aldehyde fuscin stainof a mouse islet in the mouse pancreas demonstrating the characteristicpurple granulation of healthy beta cells.

Donor Tissue Preparation

Before describing in detail specific examples of the invention, there isa brief discussion of some parameters of the invention.

Donor Tissue

In addition to permitting transplantation of islet cells, the inventioncan facilitate transplantation of any other tissue or organ, e.g.,kidney, heart, liver, lung, brain, and muscle tissue.

Antigens to be Masked, Modified, or Eliminated

The invention can be used to mask, modify, or eliminate any hostT-cell-interactive antigen on any of the cells of the donor tissue. Inaddition to HLA class I antigens, which are found on all parenchymalcells, including islet cells, other important donor cell antigens knownto interact with host T-cells to bring about rejection are LFA-3 andICAM-1; these react, respectively, with the host T-cell receptors CD2and LFA-1. Both LFA-3 and ICAM-1 are found on endothelial cells whichmake up blood vessels in transplanted organs such as kidney and heart.Masking, altering, or eliminating these antigens will facilitatetransplantation of any vascularized implant, by preventing recognitionof those antigens by CD2+ and LFA-1+ host T-lymphocytes. Further,masking, altering, or eliminating a particular donor cell antigen mayrender more than one donor cell-type less susceptible to rejection. Forexample, not only do parenchymal cells such as islet cells bear HLAclass I antigens, but passenger lymphocytes bear such antigens as well,and if such lymphocytes are present in a donor tissue preparation,removal of an HLA class I antigen or treatment of the tissue preparationwith an HLA class 1 antigen masking agent will render those lymphocytesless antigenic.

The antigens HLA class 1, LFA-3, and ICAM-1 are well-characterized, andantibodies to these antigens are publicly available, and can be made bystandard technique. For example, anti-ICAM-1 can be obtained from AMAC,Inc., Maine; hybridoma cells producing anti-LFA-3 can be obtained fromthe American Type Culture Collection, Rockville, Md.

Where the donor tissue to be transplanted bears more than oneT-cell-interactive antigen, two or more treatments, e.g., two or moremasking agents, may be used together. Alternatively, a polyclonalantisera generated against the donor tissue may be used to mask multiplecell surface antigens of the donor tissue.

Non-Lytic Masking Agents

Generally, the invention can employ three categories of masking agents:(1) antibodies or fragments or derivatives thereof; (2) solublefragments or analogs of antigen-specific host T-cell receptors; and (3)synthetic organic molecules which mimic the antigen binding propertiesof T-cell receptors.

Antibodies, the currently most preferred masking agents, can be usedeither as one or more antigen-specific preparations, or as whole donororgan or tissue antisera preparations. In either case, it is necessarythat the preparations be unable to fix complement and bring about donorcell lysis. Complement fixation can be prevented by deletion of the Fcportion of the antibody, by using an antibody isotype which is notcapable of fixing complement, or, less preferably, by using a complementfixing antibody in conjunction with a drug which inhibits complementfixation.

Individual antigen-specific antibodies can be made by standardprocedures, including immunization of an animal, e.g., a mouse, with theantigen to be masked, followed by hybridoma preparation and antibodyscreening according to standard methods. Alternatively, whole donorantisera can also be used. For example, where the donor tissue isderived from a pig, whole pig antisera are produced by immunization of amouse with pig donor tissue or pig lymphocytes, followed by screeningfor antibodies which block human T-lymphocyte adhesion to pig donorcells.

As an alternative to antibodies or antibody fragments, masking can beeffected by use of soluble host T-cell receptor which competitivelyinhibits binding of those T-cells to donor tissue cell antigens, byoccupying the antigenic site on the tissue which would otherwiseinteract with the host T-cells. T-cell molecules or proteins, e.g., CD8,CD2, and LFA-1, are well characterized proteins generally having anextracellular domain, a transmembrane region, and a cytoplasmic domainwhich bend to target cell ligands. Soluble T-cell receptor proteinfragments can be made by standard recombinant DNA procedures, in whichthe DNA encoding the transmembrane and cytoplasmic domains is deleted,and the extracellular domain DNA is expressed in recombinant cells toproduce soluble recombinant protein.

Capping

Capping is a term referring to the use of antibodies to causeaggregation and inactivation of surface antigens. First, the tissue iscontacted with an antibody specific for the antigen, so thatantigen-antibody immune complexes are formed. The next step iscontacting the tissue with a second antibody capable of forming immunecomplexes with the first antibody, so that the first antibody isaggregated to form a cap at a single location on the cell surface. Thetechnique is well known, and has been described, e.g., in Taylor et al.(1971), Nat. New Biol. 233:225-227; and Santiso et al. (1986), Blood,67:343-349. In the case of cells, e.g., islet cells, bearing HLA class Iantigens, the first step is to incubate the cells with antibody (e.g.,W6/32 antibody, described below) to HLA class I, and then to incubatewith antibody to the donor species, e.g., goat anti-mouse antibody, tobring about aggregation.

Transgenic Animals with Decreased HLA Class I Expression

As an alternative or an adjunct to masking surface antigens on cells ofdonor tissues prior to transplantation, such tissues can be grown intransgenic animals which have been genetically altered so that surfaceantigen expression is diminished. Such transgenic animals can be made bystandard transgenic techniques, employing genes which delete orinactivate the gene encoding the target antigen, or delete or inactivatea gene necessary for its expression on the cell surface, by homologousrecombination.

For example, in the case of HLA class I expression, homologousrecombination can be used either to delete or inactivate the HLA class Imolecule itself, or to inactivate or delete a companion moleculenecessary for its surface expression. The HLA class I molecule is aprotein composed of a 32Kd and a 45Kd chain, associated with anotherprotein, β-2 microglobulin. The highly conserved β-2 microglobulinprotein is believed to function as a carrier molecule which facilitatesclass I assembly in plasma membranes.

Inhibition of class I expression on the surfaces of cells, e.g., isletcells, can thus be achieved either by deletion or inactivation of one ofthe HLA class I chains, or by deletion or inactivation of the carrierβ-2 microglobulin molecule. Disruption of β-2 microglobulin expressionin transgenic animals resulting in decreased HLA class I expression hasbeen carried out by several groups (Koller and Smithies (1989), PNASU.S.A. 86:8932-8935; Zijlstra et al. (1990) Nature, 344:742-746;Doetschman et al. (1987) Nature, 51:503-512).

In Vitro Methods to Decrease HLA Class I Expression

A number of oncogenic viruses have been demonstrated to decrease HLAclass I expression in infected cells; Travers et. (1980) Int'l. Symp. onAging in Cancer, 175-180; Rees et al. (1988) Br. J. Cancer, 57:374-377.In addition, it has been demonstrated that this effect on HLA class Iexpression can be achieved using fragments of viral genomes, in additionto intact virus. Transfection of cultured kidney cells with fragments ofadenovirus causes elimination of surface HLA class I antigenicexpression; Whoshi et al. (1988) J. Exp. Med. 168:2153-2164. Forpurposes of decreasing HLA class I expression on the surfaces of donorcells, e.g., islet cells, viral fragments, which are non-infectious, arepreferable to whole viruses, which could cause complications. Otherviruses and viral fragments could be used to decrease expression ofother surface antigens on other types of donor cells, as well asdecreasing expression of HLA class I expression on parenchymal cellssuch as islet cells.

Local Blockage of Recipient T-Cell Receptors With Secreted DonorAntigens

The transplantation inhibition strategies discussed above all involvealtering the donor tissue such that surface antigens on donor tissuecells which are recognized as foreign by receptors on recipient T-cellsare eliminated, modified, or masked. An alternative strategy is tomodify the donor tissue in a different way, which brings about blockageof the host T-cell receptors by antigen secreted by donor cells. Forexample, in the case of donor tissue containing parenchymal cellsbearing surface HLA class I antigen, rather than masking the antigen,those cells can be transfected with DNA encoding soluble antigen, whichis secreted and which competitively binds to the CD8 receptor on theT-lymphocytes of the recipient which would otherwise bind tomembrane-bound HLA class I antigen on the donor tissue cells. Thetechniques for carrying out this procedure will be analogous to methodsused by other workers to bring about secretion of a recombinant proteinin concert with insulin secretion; Lo et al. (1988) Cell, 53:159-168;Adams et al. (1987) Nature, 325:223-228. Adams et al. achieved SV40 Tantigen synthesis in islet cells in concert with insulin production. HLAclass I antigen expression and secretion could be coupled to insulinproduction and secretion by placing the gene for one or both subunits ofHLA class I antigen under the control of insulin gene regulatorysequences. Insulin secretion thus will result in simultaneous expressionand secretion of HLA class I antigen. This strategy has the advantage ofcausing secretion of HLA class I antigen only from islet cells, intissue which may contain other cell types as well; none of the othercells present produce and secrete insulin. In addition, this approachconfines the soluble HLA class I antigen to the localized region whereit is needed, i.e., in the area immediately surrounding the transplantedislet cells.

The following specific examples are for illustration purposes only, andare not intended to limit the scope of the invention.

EXAMPLE 1

This example involves xenogeneic transplantation of HLA class I positivehuman islet cells into nonimmunosuppressed Balb/c mouse recipients.Freshly isolated human islets were pretreated prior to transplantationwith whole monoclonal antibody or F(ab')₂ monoclonal antibody fragmentsto conceal ("mask") donor antigens. F(ab')₂ fragments lack the Fcantibody region, thus circumventing complement-mediated killing afterantigen binding. Intact immunoglobulin was used as a control. Humanislets were treated with relevant HLA class I monoclonal antibody(W6/32) (American Tissue Culture Society) (Barnstable et al. (1978)Cell, 14:9-20) or irrelevant CD29 monoclonal antibody (CoulterCorporation, Hialeah, Fla.). Clean human islet preparations, free ofcontaminating endothelial and fibroblast overgrowth, are negative forICAM-1 expression, negative for CD29 expression, have low LFA-3expression, and are positive for HLA class I antigens (FIG. 1).Therefore, islets, unlike other cytotoxic T-lymphocyte targets, lack theprominent expression of the two important adhesion epitopes LFA-3 andICAM-1, and there is little need to protect these adhesion epitopes fromT-cell binding.

F(ab')₂ fragments were generated using an immobilized pepsin. Purifiedantibody added at 20 mg/ml in pH 4.7 digestion buffer was digested for4.5 hours for CD29 antibody and 4.0 hours for W6/32 antibody (HLA classI) at 37° C. in a shaker water bath (Pierce Chemical, Rockford, Ill.).The crude digest was removed from the pepsin and immediately neutralizedwith pH 7.0 binding buffer. This antibody mixture was applied to animmobilized Protein A column and the eluate collected for the F(ab')₂fragments. Dialysis against PBS for 24 hours using 50,000 M.W. cutofftubing was then performed to rid the digest of contaminating Fcfragments. CHAPPS, a biocompatible buffer, was added to the dialysis bagat a concentration of 10 mM. The completeness of the digest andpurification of the F(ab')₂ digests were monitored by silver staining of15% SDS gels. Final purification of the fragments was achieved by HPLCusing a Superose 12 column (Pharmacia, Upsala, Sweden).

F(ab')₂ fragments or whole antibodies were incubated with human isletsat a concentration of 1 μg of antibody for approximately 1×10⁶ isletcells for 30 minutes at room temperature. After incubation, the treatedor untreated islets were washed once in Hanks buffer containing 2% FCSand then immediately transplanted under the kidney capsule by syringeinjection. The human islets used were transplanted within 4 days afterisolation. Ten to twelve week old Balb/c female mice (The JacksonLaboratories, Bar Harbor, Me.) were transplanted with 2200-4500 humanislets. At 30 or 200 days post transplantation the mice were sacrificedby cervical dislocation and the kidney containing the transplantedtissue was surgically removed and immediately fixed in Bouin's solution.

The results of the transplantation studies are summarized in Table I.W6/32 F(ab')₂ pretreatment of donor xenogeneic islets (HLA class I)resulted in complete islet xenograft survival in all 5 recipientsevaluated at 30 days after transplantation (Group 1) as well as all 5recipients evaluated at 200 days after transplantation (Group 2). Thehistology in all 10 mice revealed well-granulated islets under thekidney capsules (FIGS. 1A, 1B). Untreated human islets were promptlyrejected by 7 days in this mouse model; histology in these mice showedmassive lymphocytic infiltrates under the kidney capsules and nogranulated islet cells. The HLA class I F(ab')₂ treated islet grafts(W6/32) were virtually free of adjacent lymphocyte deposits even at 200days following transplantation (FIG. 1B). Lymphocytic accumulations area known characteristic of xenograft transplants prolonged with culture.

Balb/c recipients receiving islet grafts pretreated with whole HLA classI W6/32 antibody demonstrated no surviving islet tissue at 30 or 200days after transplantation (Group 3, 4) (Table 1), indicating probablecomplement fixation and lysis by the whole, uncut antibody. Histologyperformed on these transplants revealed subcapsular kidney fibrosis atthe transplantation site (FIG. 1C). The coating of donor islets withirrelevant F(ab')₂ fragments directed at CD29 resulted in islet graftrejection by day 30 as well as day 200 (Group 5, 6). Intact, CD29antibody also failed to prolong islet xenograft survival (Group 7, 8).The pretreatment of donor human islets with specific HLA class I F(ab')₂antibody fragments (W6/32) and with irrelevant CD29 F(ab')₂ antibodyfragments (CD29) resulted in graft survival in all five recipients atday 30 (Group 9) and all five recipients at day 200 (Group 10) asobserved for HLA class I F(ab')₂ fragments alone. As expected, untreatedhuman islets were absent at both the 30 day and 200 day evaluation timepoints (Group 11, 12). Only subcapsular fibrosis was present under thekidney capsule at day 30 (FIG. 1C) and day 200 in these recipients.

The function of transplanted human islets was monitored by evaluatinghuman insulin C' peptide levels at 30 and 200 days post transplantation(Table 2). All 20 recipients receiving W6/32 F(ab')₂ coated human isletsor W6/32 F(ab')₂ and CD29 F(ab')₂ coated islets at day 30 had detectablehuman C' peptide levels markedly above background levels (Groups 1, 2,9, 10) (p=0.002). Human C' peptide levels were similarly detected at 200days in all ten recipients receiving W6/32 F(ab')₂ antibody coatedislets (Groups 2, 10) (p=0.003). In contrast, all individuals in thecontrol transplant groups had human C' peptide levels similar tobackground levels (Group 3, 4, 5, 6, 7, 8, 11, 12) (p=0.98).

EXAMPLE 2

This example involves the xenogeneic transplantation of rat insulinomatumor cells (RIN) into nonimmunosupressed Balb/c mouse recipients toinvestigate the possibility of graft specific tolerance with growth of atransplanted tissue pre-treated with polyclonal F(ab')₂.

RIN tumor cells are an established rat insuloma tumor cell line(Meflasson et al., 1983 J. Biol. Chem. 258:2094-2097). Polyclonal mouseanti-RIN serum was produced and F(ab')₂ antibody fragments weregenerated as described above in Example 1. As expected, xenogeneic RINcells (approx. 5,000 cells per recipient) transplanted under the kidneycapsule of nonimmunosuppressed BALB/c mice were uniformly rejected whenevaluated by histology with aldehyde fuscin staining (n=4) (Table 3). Inaddition, pretreatment of RIN cells with intact polyclonal mouseanti-RIN antibody, without removal of the complement-fixing Fc region,prior to transplantation also failed to protect grafts from recipientmediated rejection (n=4). In contrast, pretreatment of RIN cells withF(ab')₂ fragments of mouse anti-RIN polyclonal antibodies allowed RINcell survival at one, two, three, and four months after transplantation.Even though each BALB/c recipient received an equal number of cells atthe time of transplantation, serial sections through the transplant siteunder the kidney capsules at different monthly intervals aftertransplantation revealed a noticeable increase in the mass of tumortissue, suggesting tumor growth. In addition, the successfullytransplanted RIN cells demonstrated mitosis on hematoxylin and eosinstaining, confirming cell division and presumably the new expression ofunmasked foreign antigens. The continued survival and expansion of axenogeneic tumor cell line suggests the possible presence of graftinduced tolerance in the recipients. Further proof of a state of grafttolerance was obtained by transplanting F(ab')₂ coated RIN cellsunilaterally into the right kidney of nonimmunosuppressed mice for 30days prior to a secondary transplant of untreated RIN cells into theleft kidney. At day 60 the four mice transplanted in this manner weresacrificed. The four untreated secondary transplants of insulinoma cellsalso demonstrated survival, confirming the suspected development of asystemic tolerant state sufficient for fresh tumor cell survival.

EXAMPLE 3

The effectiveness of F(ab')₂ HLA class I antibody coating in preventingrejection of non-tumorgeneic human liver cells in xenogeneic transplantswas also investigated. Approximately 5,000 fresh human liver cells fromthe parenchymal tissue of the liver were injected into the subscapularspace of the kidney capsules of nonimmunosuppressed mouse recipients.Histological examination using PAS staining of the subscapular sitesindicated that all 5 transplant recipients of F(ab')₂ -treated livercells demonstrated easily located viable liver cells at the subscapularrenal site 30 days after transplantation. As expected, untreated humanliver cells were uniformly rejected in all five mice by day 30 aftertransplantation.

It is clear from the results of Examples 1 and 3 that the simpleinterruption of recipient T cell recognition by masking of foreign HLAclass I determinants allows prolonged xenograft survival up to 200 days.This new strategy eliminates recipient treatment, thus preserving theimmune response of the host so that it remains available for recognitionof relevant pathogens.

The prolonged duration of recipient unresponsiveness to a viable tissuewhich eventually might lose the masking antibody or exhibit the abilityto resynthesize new uncoated HLA class I determinants suggests thatgraft specific tolerance may stabilize these transplants. This issubstantiated by the lack of large foci of lymphocyte infiltrates in mysuccessful xenografts. This is consistent with my assumption that donorpretreatment of the graft with HLA class I antibody fragments coatsclass I antigens on transient donor dendritic cells as well as class Iantigen on the parenchymal islet cells. With the passage of timepost-transplantation, these antigen presenting cells which are potentgraft rejection initiators may die off, as occurs with extended culture,thus gradually exposing the recipient to low levels of HLA class Iantigens on non-antigen presenting cells.

OTHER EMBODIMENTS

Other embodiments are within the following claims. For example, theprocedures described above for treatment of islet cells and liver cellscan be used to treat muscle cells for transplantation into patients withmuscular dystrophy, as follows; muscle cells, like islet cells, bearrejection-stimulating HLA class I antigens, and also express class IIantigens. Human donor muscle cells will be obtained by biopsy of livingrelated donors or brain dead donors using a 14-16 gauge cutting trocharinto a 1-2 inch skin incision. The fresh muscle plug will then belightly digested into a single cell suspension using collagenase,trypsin and dispase at 37° C. Floating debris will be removed with apipet and media washes and the viable cell pellet counted aftercentrifugation at 1000 rpm×10 minutes. This cell count will then be usedto calculate the amount of HLA class I and class II antibody fragmentsto add; treatment will be as described above for islet cells. Similarly,the invention will permit transplantation of cells, from a healthyindividual or which have been genetically engineered, into recipientswho have a deficiency for a particular cellular component. For example,individuals with hemophilia might be recipients of Factor VIII-producingliver cells from normal donors, or of cells which have been geneticallyengineered to secrete Factor VIII.

Another embodiment of the invention would be the transplantation intopatients of whole organs (e.g. heart, lung, liver, kidney). A preferredorgan masking pretreatment procedure would involve perfusion of thedonor organ with F(ab')₂ fragments of monoclonal antigen-specificantibodies or with polyclonal antisera generated against the organtissue; perfusion is carried out using conventional techniques forperfusing donor organs with other fluids.

What is claimed is:
 1. A method for inhibiting rejection by a recipientanimal of a human non-lymphoid tissue which is to be transplanted into arecipient animal, said method comprising modifying, eliminating, ormasking an antigen of said non-lymphoid tissue which, when present onthe surface of a cell of said human tissue, is capable of causing aT-lymphocyte-mediated response in the recipient animal, to inhibitantigen-mediated interaction between said cell and a T-lymphocyte of therecipient animal without causing lysis of said cell.
 2. A method forinhibiting rejection by a recipient animal of a non-lymphoid tissueobtained from an animal of a different species which is to betransplanted into the recipient animal, said method comprisingmodifying, eliminating, or masking an antigen of said non-lymphoidtissue which, when present on the surface of a cell of said tissue, iscapable of causing a T-lymphocyte-mediated response in the recipientanimal, to inhibit antigen-mediated interaction between said cell and aT-lymphocyte of the recipient animal without causing lysis of said cell.3. The method of claim 1 or claim 2 wherein said inhibiting comprisesmasking said antigen by treating said tissue with a non-lytic maskingagent which is capable of forming a complex with said antigen on saidcell.
 4. The method of claim 1 or claim 2 wherein said inhibitingcomprises modifying said antigen by capping.
 5. The method of claim 1 orclaim 2 wherein said cell is an islet cell.
 6. The method of claim 1wherein said animal is a human.
 7. The method of claim 1 or claim 2wherein said masking comprises treating said tissue with a non-lyticmasking agent which comprises an antibody F(ab')₂ fragment which iscapable of forming a complex with said antigen on said cell.
 8. Themethod of claim 7 wherein said antibody is monoclonal.
 9. The method ofclaim 7 wherein said antibody comprises a polyclonal antisera againstsaid tissue.
 10. The method of claim 3 wherein said antigen is an HLAclass I antigen and a cytotoxic CD8+ lymphocyte of said animal isinhibited, by said masking agent, from interacting with said HLA class Iantigen on said cell.
 11. The method of claim 7 wherein said F(ab')₂fragment is produced by digesting intact antibody and then purifyingsaid fragment in a two-step process involving first applying thedigested antibody to a Protein A column and then further purifying thefragment by HPLC.
 12. The method of claim 3 wherein said antigen is aClass I antigen.
 13. The method of claim 12 wherein said maskingcomprises treating said tissue with a non-lytic masking agent whichcomprises at least one antibody F(ab')₂ fragment which is capable offorming a complex with said antigen on said cell.
 14. The method ofclaim 13 wherein said tissue is islets.
 15. The method of claim 12wherein said tissue is islets.
 16. The method of claim 2 wherein saidinhibiting comprises masking said antigen with a nonlytic masking agentwhich is capable of forming a complex with said antigen on said cell.17. The method of claim 16 wherein said antigen is a Class I antigen.18. The method of claim 17 wherein said masking comprises treating saidtissue with a non-lytic masking agent which comprises at least oneantibody F(ab')₂ fragment which is capable of forming a complex withsaid antigen on said cell.
 19. The method of claim 1 wherein saidantigen is a Class I antigen.
 20. The method of claim 18 wherein saidtissue is islets.